Inhibitors of fatty acid amide hydrolase

ABSTRACT

Potent inhibitors of fatty acid amide hydrolase (FAAH) are constructed having K i &#39;s below 200 pM and activities 10 2 -10 3  times more potent than the corresponding trifluoromethyl ketones. The potent inhibitors combine several features, viz.: 1.) an α-keto heterocylic head group; 2.) a hydrocarbon linkage unit employing an optimal C12-C8 chain length; and 3.) a phenyl or other π-unsaturation corresponding to the arachidonyl Δ 8,9 /Δ 11,12  and/or oleyl Δ 9,10  positions. A preferred α-keto heterocylic head group is α-keto N4 oxazolopyridine, with incorporation of a second weakly basic nitrogen. Fatty acid amide hydrolase is an enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid) and anandamide (an endogenous ligand for cannabinoid receptors).

GOVERNMENT RIGHTS

This invention was made, in part, with government support under Grantsfrom NIH, viz., Grants No. CA42056 and MH58542. The U.S. government mayhave certain rights in the invention.

TECHNICAL FIELD

The present invention relates to inhibitors of fatty acid hydrolase.More particularly, the invention relates to inhibitors of fatty acidhydrolase employing a heterocyclic pharmacophore.

BACKGROUND

Fatty acid amide hydrolase (FAAH), referred to as oleamide hydrolase andanandamide amidohydrolase in early studies, is an integral membraneprotein that degrades fatty acid primary amides and ethanolamidesincluding oleamide and anandamide, as illustrated in FIG. 1 (M. P.Patricelli, et al., (1998) Biochemistry 37, 15177-15187. D. G. Deutsch,et al., (1993) Biochem. Pharmacol. 46, 791-796; F. Desarnaud, et al.,(1995) J. Biol. Chem. 270, 6030-6035; C. J. Hillard, et al., (1995)Biochim. Biophys. Acta 1257, 249-256; N. Ueda, et al., (1995) J. Biol.Chem. 270, 23823-23827; R. L. Omeir, et al., (1995) Life Sci. 56,1999-2005; S. Maurelli, et al., (1995) FEBS Lett. 377, 82-86; and M.Maccarrone, et al., (1998). J. Biol. Chem. 273, 32332-32339). Thedistribution of FAAH in the CNS suggests that it degradesneuromodulating fatty acid amides at their sites of action and isintimately involved in their regulation (E. A. Thomas, et al., (1997) J.Neurosci. Res. 50, 1047-1052). FAAH hydrolyzes a wide range of oleyl andarachidonyl amides, the CB1 agonist 2-arachidonylglycerol, the related1-arachidonylglycerol and 1-oleylglycerol, and methyl arachidonate,illustrating a range of bioactive fatty acid amide or ester substrates.(W. Lang, et al., (1999) J. Med. Chem. 42, 896-902; S. K. Goparaju, etal., (1998) FEBS Lett. 442, 69-73; Y. Kurahashi, et al., (1997) Biochem.Biophys. Res. Commun. 237, 512-515; and T. Bisogno, et al., (1997)Biochem. J. 322, 671. Di Marzo, V., T. Bisogno, et al., (1998) Biochem.J. 331, 15-19). Although a range of fatty acid primary amides arehydrolyzed by the enzyme, FAAH appears to work most effectively onarachidonyl and oleyl substrates (B. F. Cravatt, et al., (1996) Nature384, 83-87; and D. K. Giang, et al., (1997) Proc. Natl. Acad. Sci. USA94, 2238-2242).

The important biological role of FAAH suggests a need for molecularregulators of its activity. However, only a select set of FAAHinhibitors have been disclosed. Amongst these is the potent endogenousinhibitor 2-octyl γ-bromoacetoacetate, which was discovered prior toFAAH and characterized as an endogenous sleep-inducing compound (M. P.Patricelli, et al., (1998) Bioorg. Med. Chem. Lett. 8, 613-618; and S.Torii, et al., (1973) Psychopharmacologia 29, 65-75). After thediscovery of FAAH, elaborations of 2-octyl γ-bromoacetoacetate weredeveloped and characterized as potent inhibitors of this enzyme.Moreover, subsequent inhibitors employ a fatty acid stricture attachedto pharmacophoric head group. The pharmacophoric head groups cangenerally be classified as either reversible or irreversible. Reversibleinhibitors include electrophilic carbonyl moieties, e.g.,trifluoromethyl ketones, α-halo ketones, α-keto esters and amides, andaldehydes. Irreversible inhibitors include sulfonyl fluorides andfluorophosphonates. (B. Koutek, et al., (1994) J. Biol. Chem. 269,22937-22940; J. E. Patterson, et al., (1996) J. Am. Chem. Soc. 118,5938-5945; D. L. Boger, et al., (1999) Bioorg. Med. Chem. Lett. 9,167-172; D. G. Deutsch, et al., (1997) Biochem. Pharmacol. 53, 255-260.D. G. Deutsch, et al., (1997) Biochem. Biophys. Res. Commun. 231,217-221; and L. De Petrocellis, et al., (1997) Biochem. Biophys. Res.Commun. 231, 82-88; and L. De Petrocellis, et al., (1998) In RecentAdvances Prostaglandin, Thromboxane, and Leukotriene Research, PlenumPress: New York, 259-263).

SUMMARY OF INVENTION

One aspect of the invention is directed to an inhibitor of fatty acidamide hydrolase represented by the formula A-B-C. In this formula, A isan α-keto heterocyclic pharmacophore for inhibiting the fatty acid amidehydrolase; B is a chain for linking A and C, said chain having a linearskeleton of between 3 and 9 atoms selected from the group consisting ofcarbon, oxygen, sulfur, and nitrogen, the linear skeleton having a firstend and a second end, the first end being covalently bonded to theα-keto group of A, with the following proviso: if the first end of saidchain is an α-carbon with respect to the α-keto group of A, then theα-carbon is optionally mono- or bis-functionalized with substituentsselected from the group consisting of fluoro, chloro, hydroxyl, alkoxy,trifluoromethyl, and alkyl; and C is an activity enhancer for enhancingthe inhibition activity of said α-keto heterocyclic pharmacophore, saidactivity enhancer having at least one π-unsaturation situated within aπ-bond containing radical selected from a group consisting of aryl,alkenyl, alkynyl, and ring structures having at least one unsaturation,with or without one or more heteroatoms, said activity enhancer beingcovalently bonded to the second end of the linear skeleton of B, theπ-unsaturation within the π-bond containing radical being separated fromthe α-keto group of A by a sequence of no less than 4 and no more than 9atoms bonded sequentially to one another, inclusive of said linearskeleton.

In a preferred embodiment, said α-keto heterocyclic pharmacophore isrepresented by the formula:

In the above formula, “het” is selected from the following group:

In a preferred mode of the above embodiment, “het” is selected from thefollowing group:

One group of inhibitors having a particularly high activity isrepresented by the following structure:

In the above structure, R₁ and R₂ are independently selected from thegroup consisting of hydrogen, fluoro, chloro, hydroxyl, alkoxy,trifluoromethyl, and alkyl; and “n” is an integer between 2 and 7.

Another aspect of the invention is directed to a process for inhibitinga fatty acid amide hydrolase. The process employs the step of contactingthe fatty acid amide hydrolase with an inhibiting concentration of anyof the above inhibitors represented above by the formula A-B-C.

Another aspect of the invention is directed to a process for enhancingSWS2 or REM sleep. The process employs the step of administering atherapeutically effective quantity to a patient of a fatty acid amidehydrolase inhibitors represented above by formula A-B-C.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates the catalysis of the hydrolysis reaction by fattyacid amide hydrolase (FAAH) of oleamide and anandamide substrates andtheir conversion to oleic acid and arachidonic acid, respectively.

FIG. 2 schematically illustrates alternative routes for the synthesis ofα-keto heterocyclic inhibitors of FAAH.

FIG. 3 is a table comparing the activity of α-keto heterocyclicinhibitors of FAAH with the corresponding trifluoromethyl ketoneillustrating the enhancement of activity with the addition of basicnitrogen to the heterocyclic head group.

FIG. 4 is a table illustrating the steric effects of the methyl positionof 4-, 5-, 6-, and 7-methylbenzoxazole head groups upon inhibitoryactivity with respect to FAAH catalysis and and employs this data todefine the limits to depth and width of the FAAH active site.

FIG. 5 is a table illustrating the effects of nitrogen position withinoxazolopyridine head groups with respect to inhibitory activity of FAAHcatalysis.

FIG. 6 is a table illustrating the effects of unsaturations within thehydrocarbon tail of α-keto heterocyclic inhibitors with respect toinhibitory activity of FAAH catalysis. The data shows that for C18Δ^(9,10) hydrocarbon tails, the effects of unsaturations upon inhibitoryactivity are as follows: Z (cis)>E (trans)>saturated.

FIG. 7 is a table illustrating the inhibitory activity ofarachidonyl-based α-keto heterocyclic inhibitors with respect to FAAHcatalysis. The data shows that arachidonyl-based α-keto heterocyclicinhibitors are more potent but much less stable than oleyl-based α-ketoheterocyclic inhibitors.

FIG. 8 is a table illustrating the dependence of inhibitory activityupon the length of the fatty acid side chain of α-keto heterocyclicinhibitors and upon the length of the fatty acid side chain of α-ketoheterocyclic inhibitors wherein the fatty acid side chain includes aterminal aromatic group.

FIG. 9 is a table comparing the inhibitory activity of α-hydroxyheterocycle inhibitors with that of α-keto heterocyclic inhibitors.

FIG. 10 is a table comparing the inhibitory activity of α-ketoheterocyclic inhibitors against recombinant human FAAH and rat FAAH.

FIG. 11 illustrates how the ester is functionalized at the alphaposition with fluorine, hydroxyl and trifluoromethyl groups. Anasymmetric method for making a chiral alpha-fluoro ester is given, butone familiar with the art will know how to accomplish making thetrifluoro-methyl derivative in an asymmetric fashion. These methodsassume that any functional groups that make up “R” are suitablyprotected.

FIG. 12 illustrates the methods by which chlorine,alpha-alkyl-alpha-hydroxyl, alpha-alkyl-alpha-Trifluoromethyl, andalpha-alkyl-alpha-fluoro groups may be added to an ester. Depending onwhat “R” is, some of these esters or the corresponding acids may becommercially available. A Mitsunobu reaction is done to obtain thealpha-chloro-compound from the corresponding alpha-hydroxy ester. Anasymmetric hydroxylation of an enolate of an alpha-alkyl ester isaccomplished by using an asymmetric oxaziridine (I). The last twoproducts in this figure are obtained as racemates.

FIG. 13 illustrates various structures on the leftmost portion of thepage which are just the heterocyclic portion of the alpha-ketoheterocyclic FAAH inhibitor. The alpha-keto group, which includes thetail, is not shown. The method of acylation given is either one that isdescribed in the specification or a palladium-catalyzed or anuncatalized acylation of a heteroaryl stannane with the desired acidchloride. The starting materials, along with their commercial sourcesare given in the second column. Those structures that are notcommercially available have their synthesis described in the literaturereferences listed in the third column. The remaining structures orcompounds shown in the second column may be easily synthesized bymethods known by a skilled practitioner in the art.

FIG. 14 is a continuation of FIG. 13 and shows more monocyclicheterocycles and their methods of acylation, starting materials andliterature references describing their syntheses. Where no method ofacylation is given, it is contained within the references for thesynthesis for that particular heterocycle. The use of an aryl stannanewith the acid chloride under palladium-catalyzed cross-coupling reactionis assumed and a literature reference for this reaction was given inFIG. 13.

FIG. 15 lists the bicyclic heterocycles synthesized, the requiredmethods of acylation, the starting material required, and literaturereferences for synthesizing the desired heterocycles. Two methods ofsynthesizing the oxazolo[4,5-d]pyridazines are given as one may besuperior for a given tail segment or functionality.

FIG. 16 is a continuation of FIG. 15 and the method of acylation wasgiven in the specification.

FIG. 17 is a continuation of FIG. 16 and illustrates methods ofacylation, starting materials, and literature references for theremaining two 5,6 bicycloheteroaromatic compounds. Here, the method ofacylation is more complex as it is stepwise. This is necessary becauseof the complex ring and lower stability of these compounds.

FIG. 18 is a continuation of FIG. 17 and illustrates methods ofacylation, starting materials, and literature references for 5,5heteroaromatic compounds. The first compound may be obtained by themethod given in the reference in column three. The starting materialsare commercially available. The last type of heterocycle is a 5,5heteroaromatic system that has seen much commercial application.

FIG. 19 illustrates in chart form oligoethylene glycol chains which arecommercially available. The acylation of t-butyl bromoacetate isobtained as shown and and the hydrolysis of t-butyl esters is easilyachieved in acidic solution to give the free acid.

FIGS. 20-22 illustrate a partial list of commercially availablehydrocarbon acids (from Aldrich (TM) catalog).

DETAILED DESCRIPTION

An unusually potent class of competitive inhibitors of FAAH wasdeveloped based on the additive, complementary binding interactionsprovided by the electrophilic carbonyl of an α-keto heterocycle and thatof the heterocycles (e.g., oxazolopyridines) including a weakly basicnitrogen and other factors. The heterocycles are not spaciallyconstrained and likely constitute an interaction with a mobile, activesite residue intimately involved in the catalysis of the amide bondcleavage reaction. FAAH belongs to a new class of amidases that have notbeen extensively studied and appear to possess a distinct combination ofactive site residues involved in catalysis. Mutagenesis studies havecharacterized it as a possible serine-lysine type amidase which lacks aparticipating active site histidine (M. P. Patricelli, et al., (1999)Biochemistry, 38(43):14125-14130). It utilizes an active site serinenucleophile (Ser 241) and incorporates two additional active siteserines (Ser 217 and 218) that enhance catalysis presumably by assistingproton transfer (M. P. Patricelli, et al., (1999) Biochemistry 38,9804-9812). Key to its enhanced amide versus ester bond cleavage is itsenlistment of a lysine (Lys 142) with a strongly perturbed pKa (7.8) asa base for Ser 241 deprotonation and which functions as a subsequentacid for protonation of the amine leaving group (M. P. Patricelli, etal., (1999) Biochemistry, 38(43): 14125-14130). It is possible that theimpact of the second, weakly basic nitrogen of the oxazolopyridines isderived from its hydrogen-bonding to one or more of these active sitesresidues and that the positioning of this residue is sufficientlyflexible as to interact with a weakly basic nitrogen in a range oflocations.

A number of well-defined relationships were observed in the developmentof the potent inhibitors. Several classes of oleyl α-keto heterocyclesexhibit FAAH inhibition comparable to the corresponding α-keto ester andcarboxamide. The more potent include 6-membered heterocyclesincorporating a second, weakly basic nitrogen as well as benzoxazole.Substitution at any of the available sites on the α-keto benzoxazoleinhibitor (C4-C7) eliminated activity defining limits to the depth andwidth of the FAAH active site Incorporation of an additional basicnitrogen into the benzoxazole skeleton providing the four isomericoxazolopyridines (N4-N7), afforded exceptionally potent FAAH inhibitors50-200 times more active than the benzoxazole and 8-40 times more activethan the corresponding trifluoromethyl ketone. Arachidonyl-basedinhibitors were found to be 2-3 times more potent than the oleyl-basedinhibitors consistent with the relative rates of FAAH hydrolysis ofanandamide versus oleamide, but are sufficiently unstable as to precludetheir effective use. The removal of the oleyl Δ^(9,10) cis double bondor the incorporation of a trans olefin reduced inhibitor potencyconsistent with prior observations (J. E. Patterson, et al., (1996) J.Am. Chem. Soc. 118, 5938-5945; and D. L. Boger, et al., (1999) Bioorg.Med. Chem. Lett. 9, 167-172). The inhibitor potency exhibited a smoothdependency on the fatty acid chain length,C18<C16<C14<C12-C8>C7>C6>C5>C2, exhibiting the maximum potency at C12-C8which corresponds to the location of the oleyl Δ^(9,10) cis double bondand the arachidonyl Δ^(8,9)/Δ^(11,12) double bonds. This appears tocorrespond to the location of a conformational bend, but not hairpinconformation, in the bound conformation identified withconformationally-restained inhibitors (D. L. Boger, et al., (1999)Bioorg. Med. Chem. Lett. 9, 167-172). This indicates that the C1-C8carbons of the inhibitors or substrates contribute substantially andincrementally to binding, and that the C14-C18/C20 carbons may actuallydiminish binding. Incorporation of π-unsaturation into the medium length(C12-C8) inhibitors at the sites of oleyl or arachidonyl unsaturationfurther enhances the inhibitor potency and this may be accomplished withsimple incorporation of a phenyl ring. The combination of thesefeatures: C8-C12 chain length, phenyl ring incorporation at thearachidonyl Δ^(8,9) and oleyl Δ^(9,10) location, and an α-ketoN4-oxazolopyridine provides FAAH inhibitors with potencies that dropbelow K_(i)'s of 200 pM being 10²-10³ times more potent than thecorresponding trifluoromethyl ketones. With these potent inhibitors, theremoval of the keto group reduces potency >10⁵ times and its reductionto an alcohol reduces potency 10³ times. The α-hydroxy oxazolopyridines,while being 10³ times less potent than the corresponding ketones,exhibit effective FAAH binding and inhibition comparable to many of theinitial α-keto heterocycles or related α-keto ester and carboxamideinhibitors. This indicates that there are complementary and significantindependent active site interactions of the α-hydroxy group and theheterocycle. The interaction of the second basic nitrogen likelyinvolves a mobile active site residue involved in the catalysis of amidebond cleavage and would be consistent with hydrogen-bonding to theactive site nonnucleophile serines or catalytic lysine.

Methods

Inhibitor Synthesis:

The α-keto heterocycles were prepared directly by addition of theheteroaryl lithium reagent to the Weinreb amide (Method A), orindirectly from the aldehyde proceeding through the α-hydroxyheterocycles followed by Dess-Martin oxidation via addition of theheteroaryl lithium reagent (Method B) or by cyanohydrin formation,acid-catalyzed conversion to the imidate (HCl-EtOH, CHCl₃), andcondensation with a 2-aminoalcohol (oxazoline), 2-aminoaniline(benzimidazole), 2-aminophenol (benzoxazole), or o-amino-hydroxypyridine(oxazolopyridine) (Method C), FIG. 2. Full details of the inhibitorsynthesis and characterization are provided in supplementary material.

Inhibition Studies:

All enzyme assays were performed at 20-23° C. using a solubilized liverplasma membrane extract containing FAAH in a reaction buffer of 125 mMTris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM HEPES, pH 9.0buffer (M. P. Patricelli, et al., (1998) Bioorg. Med. Chem. Lett. 8,613-618; and J. E. Patterson, et al., (1996) J. Am. Chem. Soc. 118,5938-5945). The initial rates of hydrolysis were monitored by followingthe breakdown of ¹⁴C-oleamide to oleic acid as previously described (B.F. Cravatt, et al., (1995) Science 268, 1506-1509; and M. P. Patricelli,et al., (1998) Bioorg. Med. Chem. Lett. 8, 613-618). The inhibition wasreversible, non time-dependent and linear least squares fits were usedfor all reaction progress curves and R² values were consistently >0.97.IC₅₀ values were determined from the inhibition observed at 3-5different inhibitor concentrations (from three or more trials at eachinhibitor concentration) using the formula IC₅₀=[I]/[(K₀/K_(i))−1],where K₀ is the control reaction rate without inhibitor and K_(i) is therate with inhibitor at concentration [I] (K. Conde-Frieboes, et al.,(1996) J. Am. Chem. Soc. 118, 5519-5525). K_(i) values were determinedby the Dixon method (x-intercepts of weighted linear fits of [I] versus1/rate plots at constant substrate concentration, which were convertedto K_(i) values using the formula K_(i)=−x_(int)/[1+[S]/K_(m)]).Previous work demonstrated the rat and human enzyme are very homologous(84%), exhibit near identical substrate specificities, and incorporatean identical amidase consensus sequence and SH3 binding domainsuggesting the observations made with rat FAAH will be similar if notidentical to those of human FAAH (B. F. Cravatt, et al., (1996) Nature384, 83-87; and D. K. Giang, et al., (1997) Proc. Natl. Acad. Sci. USA94, 2238-2242).

EXAMPLES

Nature of the Heterocycle:

A wide range of five- and six-membered monocyclic heterocycles and thethree most prevalent bicyclic heterocycles (benzthiazole, benzimidazole,and benzoxazole) were incorporated into the oleyl α-keto heterocycles8-24. The results of their examination are summarized in FIG. 3 alongwith the comparison data for the trifluoromethy ketone 3 and the relatedinhibitors 4-7 (J. E. Patterson, et al., (1996) J. Am. Chem. Soc. 118,5938-5945). The inhibitors contain the oleyl chain possessing the 9-Zdouble bond and a carbonyl at the site of the oleamide carboxamide andadjacent to the electron-deficient heterocycle. Although, many of theinhibitors were more potent than oleyl aldehyde (4) and comparable tothe α-keto ester 6 and carboxamide 7, only two, 14 and 10, matched thepotency of the trifluoromethyl ketone 3. Many of the observations madeby Edwards on the relative potencies of the α-keto heterocycles againstelastase were also observed with FAAH. This includes the unique potencyof the benzoxazole versus benzthiazole and benzimidazole, the morepotent activity of the oxazole 10 versus the thiazole or imidazole, andthe substantially more potent behavior of the 2-methyl versus 1-methyltetrazoles 14 and 13. In contrast to the observations of Edwards andunique to the studies with FAAH, the oxazole 10 proved substantiallymore potent than the oxazoline 11, and the six-membered heterocyclescontaining two nitrogen atoms, one of which remains weakly basic (17-19versus 20), were unusually potent exceeding the activity of the α-ketoester and carboxamide 7 and 8 and approaching that of trifluoromethylketone 3. Although there are many potential explanations for thisbehavior, one that was explored and proved consistent with subsequentobservations is the enhancement of the inhibitor potency byincorporation of a weakly basic nitrogen.

Steric Requirements Surrounding the Benzoxazole:

The benzoxazole 23 was chosen for further examination since it providedthe greatest opportunity for further functionalization. The 4-, 5-, 6-,and 7-methylbenzoxazoles were examined to define substitution sitesavailable for functionalization without adversely affecting theinhibitor potency, FIG. 4. Substitution of any available position on thebenzoxazole results in a greatly diminished (28) or complete loss ofactivity (25-27). This defines very precise limits to the size and depthof the FAAH active site which in turn has predictable implications onits substrate specificity or selectivity.

Oxazolopyridines: Incorporation of Nitrogen into the Benzoxazole:

Based on the observation that incorporation of an additional basicnitrogen into the heterocycles seemed to correlate with enhancedinhibitor potency, the four possible oxazolopyridines 29-32 wereexamined and found to be substantially more potent FAAH inhibitors, FIG.5. The introduction of a nitrogen into the benzoxazole skeleton enhancedthe potency 50-200 times providing inhibitors that are 10-50 times morepotent than the trifluoromethyl ketone 3. Although N4 incorporationprovided the most potent inhibitor 29, N5-N7 incorporation also providedeffective inhibitors (N4>N6>N5>N7) and there is only a 4-5 folddifference in the most and least potent agent in the series. Although itis tempting to invoke an active site interaction that uniquely involvesa dual interaction with N3 and N4, the comparable activity of 29-32suggests the interaction of the second nitrogen is more flexible.

Since Edwards disclosure of α-keto heterocycles as effective proteaseinhibitors, a number of protease inhibitors have been disclosed based onanalogous design principles (P. D. Edwards, et al., (1992) J. Am. Chem.Soc. 114, 1854-1863; P. D. Edwards, et al., (1995) J. Med. Chem. 38,76-85. Edwards, P. D., et al., (1995) J. Med. Chem. 38, 3972-3982; S.Tsutsumi, et al., (1994) Bioorg. Med. Chem. Lett. 4, 831-834. S.Tsutsumi, et al., (1994) J. Med. Chem. 37, 3492-3502; M. J. Costanzo, etal., (1996) J. Med. Chem. 39, 3039-3043; Y. Akiyama, et al., (1997)Bioorg. Med. Chem. Lett. 7, 533-538; S. Y. Tamura, et al., (1997)Bioorg. Med. Chem. Lett. 7, 1359-1364; W. Ogilvie, et al. (1997) J. Med.Chem. 40, 4113-4135; P. D. Boatman, et al., (1999) J. Med. Chem. 42,1367-1375; and R. J. Cregge, et al., (1998) J. Med. Chem. 41,2461-2480). The design principles developed by Edwards and others withregard to α-keto heterocyclic protease inhibitors may be employed incombination with the design principles disclosed herein with regard toα-keto heterocyclic FAAH inhibitors to achieve elevated potencies wellbeyond that achieved by simple introduction of the electrophiliccarbonyl.

Impact of the Double Bond:

The importance of the oleyl double bond was examined with three of theinitial potent inhibitors, FIG. 6. Identical to observations made withboth the trifluoromethyl ketone and α-keto ester FAAH inhibitors, 29 and17 containing the cis double bond were more potent than 33 and 35,respectively, containing the trans double bond which in turn were morepotent than 34 and 36 in which the double bond was removed. Similarly,23 was more potent than 37 and these results parallel those seen withthe trifluoromethyl ketone inhibitors (J. E. Patterson, et al., (1996)J. Am. Chem. Soc. 118, 5938-5945; and D. L. Boger, et al., (1999)Bioorg. Med. Chem. Lett. 9, 167-172).

Arachidonyl-Based Inhibitors:

Since the two best substrates for FAAH are arachidonamide and oleamide,five of the potent α-keto heterocycles incorporated into the arachidonyland compared to the analogous compounds having an oleyl skeleton, FIG. 7(D. K. Giang, et al., (1997) Proc. Natl. Acad. Sci. USA 94, 2238-2242)In each instance, the inhibitors were unstable and decomposed fairlyrapidly under typical working conditions. Several proved too unstable topurification to accurately assess their inhibitor potency and that of 40could only be approximated (ca. 50% purity). Where this could beaccurately assessed, the arachidonyl α-keto heterocycle inhibitors were2-5 times more potent than the oleyl-based inhibitor. Despite thisenhancement, which is consistent with the FAAH substrate preference forarachidonamide versus oleamide (rel. rate of hydrolysis 1:0.7), theirinstability precludes effective utility.

In studies with conformationally restricted trifluoromethyl ketoneinhibitors, a well-defined trend favoring a bound bent, but not hairpin,conformation was observed and defined the shape characteristics of theactive site (D. L. Boger, et al., (1999) Bioorg. Med. Chem. Lett. 9,167-172). The enhanced potency of the arachidonyl-based inhibitors islikely to be related to this shape characteristic of the FAAH activesite and their enhanced preference for adoption of the required boundconformation.

The Fatty Acid Chain:

Well-behaved trends were observed in exploring modifications in thefatty acid chain, FIG. 8. A very well-defined effect of the chain lengthwas observed and the greatest potency was found with saturated straightchain lengths of C12-C8. This is a chain length that terminates at thelocation of the Δ^(9,10) double bond of oleamide and theΔ^(8,9)/Δ^(11,12) double bond of arachidonamide and corresponds thelocation of the bend in the bound conformation identified in studieswith trifluoromethyl ketone inhibitors (D. L. Boger, et al., (1999)Bioorg. Med. Chem. Lett. 9, 167-172). Thus, the inhibitor potencyprogressively increased as the chain length was shortened from C18 toC12 (K_(i), 11 (0.6 nM), leveled off at C12-C8 with subnanomolar K_(i)'s(0.57-0.73 nM), and subsequently diminished sharply as the chain lengthwas further shortened from C8 to C2 ultimately providing inactiveinhibitors (K_(i)=0.7 (>100,000 nM). This indicates that each of thefirst C1-C8 carbons in the chain contribute significantly to inhibitorand substrate binding and that C10-C12 contribute nominally to binding.More importantly, it indicates that the terminal carbons of the longerC14-C18 inhibitors may actually diminish inhibitor binding affinity andmay not be involved in substrate binding.

Incorporating unsaturation into the fatty acid chain increases inhibitorpotency and its most effective incorporation examined proved to be thatof a benzene ring, FIG. 8. This provided inhibitors with subnanomolarK_(i)'s with the most potent inhibitor 53 possessing a K_(i) lower than200 pM, below which an accurate K_(i) could not be established in thepresent assay. This extraordinary potency was observed with thestructurally simple inhibitors 51-53 readily amendable to furthermodification. These observations, like those of the straight chaininhibitors 42-50, are analogous to those made with a series oftrifluoromethyl ketone inhibitors (D. L. Boger, et al., (1999) Bioorg.Med. Chem. Lett. 9, 167-172.). The distinction being that the α-ketooxazolopyridine inhibitors are 10²-10³ times more potent than thecorresponding trifluoromethyl ketones.

The Electrophilic Carbonyl:

Key to the design of the inhibitors was the electrophilic carbonyl whichis required for potent enzyme inhibition. A select set of the α-hydroxyprecursors to the initial inhibitors were examined and typically provedinactive as FAAH inhibitors, FIG. 9. Significantly, the α-hydroxyprecursors 59 and 60 to the potent α-keto oxazolopyridines 29 and 44,respectively, retained significant FAAH inhibition with K_(i)'s of 1.8and 1.2 μM, respectively. Although this is approximately 10³ times lesspotent than the corresponding keto derivative, they approximate thepotency of the initial series of α-keto heterocycles and that of 4-7(FIG. 3) including the oleyl aldehyde, α-keto ester, and α-ketocarboxamide. This indicates that the pyridine nitrogen of the N4oxazolopyridine, and presumably that of the N5-N7 oxazolopyridines, inconjunction with the α-hydroxy group contributes substantially to FAAHactive site binding independent of the contributions of theelectrophilic carbonyl. The corresponding agents 61 and 62 furtherlacking the α-hydroxy groups were inactive thereby losing an additional10² fold binding affinity with removal of the alcohol or 10⁵ foldbinding affinities with respect to removal of the keto group.

Therapeutic Activity:

The in vivo properties of the inhibitors detailed herein and theiraction on the identified oleamide and anandamide potential sites ofaction are presented herein. A study with 17 with only 4 treated animals(10 mg/kg ip) versus controls revealed that within the first 4 h ofadministration, 17 decreased the time spent in wakefulness by 14% of thetotal time (20% reduction) and increased the time spent in SWS2 (10%increase of the total time, 45% increase) and REM sleep (4% increase oftotal time, 75% increase). Accordingly, the inhibitors disclosed hereinare useful as inhibitors of FAAH and related amidases, and as atherapeutic agents with applications as sleep aids or analgesics whichact by preserving endogenous levels of oleamide and anandamide.

Synthetic Protocols:

1-Oxo-1-(2-thiazolyl)-9(Z)-octadecene (8). Method A1:

A modification of the method of P. D. Edwards et al. was employed(Edwards, P. D., et al., (1995) J. Med. Chem. 38, 7685). A solution ofthiazole (13.0 mg, 0.154 mmol, 1 equiv) in anhydrous THF (3.8 mL) at 30°C. was treated dropwise with n-BuLi (2.5 M in hexanes, 0.061 mL, 0.154mmol, 1 equiv) under N₂ and the mixture was stirred for 30 min. Asolution of the Weinreb amide of oleic acid (S1,N-methoxy-N-methyl-9(Z)-octadecenamide)3 (50.0 mg, 0.154 mmol, 1 equiv)in anhydrous THF (1 mL) was added rapidly, and the mixture was stirredfor 30 min at 30° C. Saturated aqueous NaCl (10 mL) was added and themixture was extracted with Et₂O (3 10 mL). The organic layers were dried(Na₂SO₄), filtered, and evaporated. Chromatography (SiO₂, 1.5 22 cm,10-20% EtOAchexanes gradient elution) afforded 8 (20.0 mg, 37%) as acolorless oil.

1-(1-Methylimidazol-2-yl)-1-oxo-9(Z)-octadecene (9): This material wasprepared in 66% yield from S1 and 1-methylimidazole using the proceduredescribed as Method A1.

1-(1-Methylbenzimidazol-2-yl)-1-oxo-9(Z)-octadecene (22): This materialwas prepared in 62% yield from S1 and 1-methylbenzimidazole using theprocedure described as Method A1.

1-(2-Benzothiazolyl)-1-oxo-9(Z)-octadecene (24): This material wasprepared in 65% yield from S1 and benzothiazole using the proceduredescribed as Method A1.

1-Oxo-1-(2-pyrazinyl)-9(Z)-octadecene (19). Method A2 (Ple, N., et al.,(1995) J. Org. Chem. 60, 37813786): A solution of2,2,6,6-tetramethylpiperidine (0.208 mL, 1.23 mmol, 4.0 equiv) inanhydrous THF (6.8 mL) at 30° C. was treated dropwise with n-BuLi (1.6 Min hexanes, 0.768 mL, 1.23 mmol, 4.0 equiv) under N₂. The reactionmixture was warmed to 0° C. and allowed to stir for 30 min. The reactionmixture was then cooled to 78° C., a solution of pyrazine (26.0 mg,0.308 mmol, 1 equiv) in anhydrous THF (1 mL) was added, and then asolution of S1 (100.0 mg, 0.308 mmol, 1 equiv) in anhydrous THF (0.5 mL)was added. After the mixture was stirred for 1 h at 78° C., a mixture of12 N HCl/THF/EtOH (1:4.5:4.5) (20 mL) was added and the reaction mixturewas slowly warmed to 25° C. Saturated aqueous NaHCO₃ (20 mL) was addedand the mixture was extracted with CH₂Cl₂ (3 20 mL). The organic layerswere dried (Na₂SO₄), filtered, and evaporated. Chromatography (SiO₂, 1.525 cm, 10-40% EtOAchexanes gradient elution) afforded 19 (13.0 mg, 12%)as a yellow oil.

1-Oxo-1-(2-pyridyl)-9(Z)-octadecene (16): This material was prepared in76% yield from S1 and 2-bromopyridine using the procedure described asMethod A2.

1-Oxo-1-(3-pyridazinyl)-9(Z)-octadecene (17): This material was preparedin 11% yield from S1 and pyridazine using the procedure described asMethod A2. 17; mp 4042° C.

1-Oxo-1-phenyl-9(Z)-octadecene (15). Method A3: A solution of S1 (111.1mg, 0.341 mmol, 1 equiv) in anhydrous THF (1 mL) at 0° C. was treateddropwise with phenylmagnesium bromide (1.0 M in THF, 0.68 mL, 0.680mmol, 2 equiv) under N₂ and stirred for 1 h. Cold-water (1 mL) was addedslowly and the resulting mixture was extracted with EtOAc (3 15 mL) andwashed with H₂O (20 mL). The organic layers were dried (Na₂SO₄),filtered, and evaporated. Chromatography (SiO₂, 1.5 20 cm, 2%EtOAchexanes) afforded 15 (97.4 mg, 83%) as a colorless oil.

N-Methoxy-N-methyl-9(E)-octadecenamide (S2). A solution of elaidic acid(1.0 g, 3.54 mmol, 1 equiv) in anhydrous CH₂Cl₂ (17 mL) at 0° C. wastreated dropwise with oxalyl chloride (2 M in CH₂Cl₂, 5.25 mL, 10.5mmol, 2.97 equiv) under N₂. The reaction mixture was allowed to warm to25° C. and stirred for 3 h. The solvent was evaporated to afford thecrude carboxylic acid chloride. Excess N,O-dimethylhydroxylamine inEtOAc (the hydrochloride salt was extracted into EtOAc from a 50%aqueous NaOH solution before use) was added slowly to the ice-cold crudematerial. The reaction mixture was stirred for 1 h, quenched with theaddition of H₂O (20 mL), and extracted with EtOAc (3 15 mL). The organiclayers were dried (Na₂SO₄), filtered, and evaporated. Chromatography(SiO₂, 2.5 20 cm, 30-60% EtOAchexanes gradient elution) afforded S2(0.96 g, 83%) as a colorless oil.

1-Oxo-1-(3-pyridazinyl)-9(E)-octadecene (35): This material was preparedin 40% yield from S2 and pyridazine using the procedure described asMethod A2.

N-Methoxy-N-methyl-octadecanamide (S3): This material was prepared in99% yield from octadecanoic acid using the procedure described above forS2. S3; mp 3234° C.

1-Oxo-1-(3-pyridazine)octadecane (36): This material was prepared in 12%yield from S3 and pyridazine using the procedure described as Method A2.36; mp 8385° C.

N-Methoxy-N-methyl-arachidonamide (S4): This material was prepared in96% yield from arachidonic acid using the procedure described above forcompound S2.

1-(3-Pyridazinyl)arachidonaldehyde (41): This material was prepared in32% yield from S4 and pyridazine using the procedure described as MethodA2.

1-Oxo-1-(4-pyrimidyl)-9(Z)-ocadecene (18). Method B:

1-Hydroxy-1-(4-pyrimidyl)-9(Z)-octadecene: This material was prepared in9% yield from the aldehyde and pyrimidine using the procedure describedas Method A2.

1-Oxo-1-(4-pyrimidyl)-9(Z)-octadecene (18): A solution of the alcohol(22.0 mg, 0.0635 mmol, 1 equiv) in anhydrous CH₂Cl₂ (7 mL) was treatedwith DessMartin's periodinane (46.0 mg, 0.109 mmol, 1.71 equiv). Thereaction mixture was stirred at 25 C for 3 h. A mixture of 10% aqueousNa₂S₂O₃/saturated aqueous NaHCO₃ (1:1) (20 mL) was added and thereaction mixture was stirred for 10 min. The reaction mixture wasextracted with CH₂Cl₂ (3 15 mL). The organic layers were dried (Na₂SO₄),filtered, and evaporated. Chromatography (SiO₂, 1.5 20 cm, 1%MeOHCH₂Cl₂) afforded 18 (17.3 mg, 79%) as a colorless oil.

1-Hydroxy-1-(1-benzyloxymethyl-1H-tetrazol-5-yl)-9(Z)-octadecene: Thismaterial was prepared in 57% yield from oleyl aldehyde and1-benzyloxymethyl-1H-tetrazole using the procedure described as MethodA1.

1-Hydroxy-1-(1H-tetrazol-5-yl)-9(Z)-octadecene: A solution of theaddition product above (59.1 mg, 0.129 mmol, 1 equiv) in 1,4-dioxane (3mL) at 25° C. was treated with 12 N HCl (3 ml, 36 mmol, 280 equiv) andstirred for 1 h. H₂O (15 mL) was added to the reaction mixture. Themixture was extracted with EtOAc (3 20 mL) and washed with saturatedaqueous NaCl (20 mL). The organic layers were dried (Na₂SO₄), filtered,and evaporated. Chromatography (SiO₂, 1.5 20 cm, 05% MeOHEtOAc gradientelution) afforded the product (40.5 mg, 93%) as a white solid; mp 7576°C.

1-Hydroxy-1-(2-methyl-2H-tetrazol-5-yl)-9(Z)-octadecene and

1-Hydroxy-1-(1-methyl-1H-tetrazol-5-yl)-9(Z)-octadecene: A suspension ofthe precursor alcohol (10.0 mg, 0.0297 mmol, 1 equiv) in anhydrous DMF(0.5 mL), MeI (6.0 uL, 0.0964 mmol, 3.25 equiv), and K₂CO₃ (8.3 mg,0.060 mmol, 2.02 equiv) was stirred for 30 min at 0° C. The reactionmixture was warmed to 25° C. and stirred for 16 h. H₂O (10 ml) was addedto the reaction mixture. The mixture was extracted with Et₂O (3 15 mL)and washed with H₂O (10 mL). The organic layers were dried (Na₂SO₄),filtered, and evaporated. Chromatography (SiO₂, 1.5 17 cm, 2%MeOHCH₂Cl₂) to afford the alcohol precursors to 14 (1.8 mg, 17%) and 13(2.7 mg, 26%) and their mixture (4.9 mg, 47%) as colorless oils.

1-(2-Methyl-2H-tetrazol-5-yl)-1-oxo-9(Z)-octadecene (14): This materialwas prepared in 67% yield from the alcohol using the procedure describedfor 18.

1-(1-Methyl-1H-tetrazol-5-yl)-1-oxo-9(Z)-octadecene (13): This materialwas prepared in 34% yield from the alcohol using the procedure describedfor 18.

1-Oxazolo-1-oxo-9(Z)-octadecene (10): A solution of oxazole (39 mg, 0.56mmol, 1.0 equiv) in anhydrous THF (6.0 mL) at −78° C. was treateddropwise with n-BuLi (2.5 M in hexanes, 0.340 mL, 0.85 mmol, 1.4 equiv)under N₂ and the resulting solution was stirred at −78° C. for 20 min.ZnCl₂ (0.5 M in THF, 2.260 mL, 1.13 mmol, 2.0 equiv) was added to themixture, and the mixture was warmed to 0° C. The mixture was stirred at0° C. for 45 min, before CuI (107 mg, 0.56 mmol, 1.0 equiv) was added tothe mixture. The mixture was stirred at 0° C. for 10 min, before asolution of 9(Z)-octadecenyl chloride (prepared from 320 mg of oleicacid and 432 mg of oxalyl chloride, 1.13 mmol, 2.0 equiv) in anhydrousTHF (11 mL) was added dropwise and the mixture was stirred at 0° C. foran additional 1 h. The reaction mixture was diluted with EtOAc (30 mL)and washed with 1:1 NH₄OH-water (20 mL), H₂O (20 mL) and saturatedaqueous Naa (20 mL), successively. The organic layer was dried (Na₂SO₄),filtered, and evaporated. Column chromatography (SiO₂, 2.4 (10 cm, 5-10%Et₂O-hexanes gradient elution) afforded 1-oxazolo-1-oxo-9(Z)-octadecene(49.1 mg, 0.15 mmol, 26% yield) as a pale yellow oil.

The reaction conditions are reported to provide C-2 regioselectiveacylation of oxazole and its derivatives (Harn, N. K., et al., (1995)Tetrahedron Lett. 36, 9453-9456). The chemical shifts of the oxazoleprotons of the product (¹H NMR) confirmed that it is the desired C-2acylated oxazole, compared with those of potential regioisomers (Hodges,J. C., et al., (1991) J. Org. Chem. 56, 449-452; and Edwards, P. D., etal., (1995) J. Med. Chem. 38, 76-85).

1-(2-Oxazolinyl)-1-oxo-9(Z)-octadecene (11). Method C1: A solution ofoleyl aldehyde (1.14 g, 4.27 mmol, 1 equiv) in THF (15 mL) and H₂O (16.5mL) was treated with KCN (2.81 g, 43.2 mmol, 10.1 eq). The reactionmixture was stirred at 25° C. for 72 h. H₂O (20 mL) and Et₂O (20 mL)were added to the reaction mixture. The mixture was extracted with Et₂O(3 20 mL) and washed with saturated aqueous NaHCO₃ (20 mL) and saturatedaqueous NaCl (20 mL). The organic layers were dried (Na₂SO₄), filtered,and evaporated to afford the cyanohydrin (1.26 g, quant.) as an oilwhich was used without further purification.

A solution of anhydrous EtOH (0.76 mL, 12.9 mmol, 20.0 equiv) in CHCl₃(1 mL) at 0 C was treated with acetyl chloride (0.74 mL, 10.4 mmol, 16.1equiv) followed by a solution of the cyanohydrin (189.6 mg, 0.646 mmol,1 equiv) in CHCl₃ (2 mL). The reaction mixture was allowed to warm to 25C and stirred for 13 h. The solvent was evaporated to afford the imidateas a white solid which was used without further purification.

A solution of the imidate in anhydrous CH₂Cl₂ (3 mL) was treated withethanolamine (78 μL, 1.29 mmol, 2.0 equiv) and triethylamine (180 μL,1.29 mmol, 2.0 equiv). The reaction mixture was stirred at 25 C for 22h. Et₂O (20 mL) and H₂O (20 mL) were added and the reaction mixture wasextracted with Et₂O (3 20 mL), and washed with saturated aqueous NaCl(20 mL). The organic layers were dried (Na₂SO₄), filtered, andevaporated. Chromatography (SiO₂, 1.5 18 cm, 7580% EtOAchexanes gradientelution) afforded the alcohol (72 mg) as a colorless oil.

A solution of the alcohol (72 mg) in anhydrous CH₂Cl₂ (5 mL) was treatedwith DessMartin's periodinane (152.3 mg, 0.36 mmol). The reactionmixture was stirred at 25 C for 70 min. Et₂O (20 mL) and saturatedaqueous Na₂S₂O₃/saturated aqueous NaHCO₃ (1:1) (20 mL) were added andreaction mixture was stirred for 10 min. The mixture was extracted withEt₂O/EtOAc (2:1) (3 30 mL) and washed with saturated aqueous NaHCO₃ (20mL) and saturated aqueous NaCl (20 mL). The organic layers were dried(Na₂SO₄), filtered, and evaporated. Chromatography (SiO₂, 1.5 18 cm,CH₂Cl₂) afforded 11 (40.7 mg, overall 18%) as a colorless oil.

1-(2-Benzoxazolyl)-1-hydroxy-9(Z)-octadecene (57). Method C2: Amodification of the method of P. D. Edwards et al. was employed(Edwards, P. D., et al., (1995) J. Med. Chem. 38, 7685). A solution ofanhydrous EtOH (0.42 mL, 7.16 mmol, 20.4 equiv) in CHCl₃ (1 mL) at 0 Cwas treated with acetyl chloride (0.40 mL, 5.63 mmol, 16.0 equiv)followed by a solution of oleyl aldehyde cyanohydrin (103.0 mg, 0.351mmol, 1 equiv) in CHCl₃ (1 mL). The reaction mixture was allowed to warmto 25 C and stirred for 16 h. The solvent was evaporated to afford theimidate as a white solid which was used without further purification. Asolution of the imidate in EtOH (2 mL) was treated with 2-aminophenol(39.4 mg, 0.36 mmol, 1.03 equiv). The reaction mixture was heated to 60C for 4.5 h. Et₂O (10 mL) and 1 N aqueous NaOH (10 mL) were added andthe reaction mixture was extracted with EtOAc/Et₂O (2:1) (3 20 mL), andwashed with saturated aqueous NaCl (20 mL). The organic layers weredried (Na₂SO₄), filtered, and evaporated. Chromatography (SiO₂, 1.5 20cm, CH₂Cl₂) afforded the product alcohol 57 (69.4 mg, 51%) as a paleyellow oil.

1-Hydroxy-1-(4-methylbenzoxazol-2-yl)-9(Z)-octadecene: This material wasprepared in 47% yield from oleyl aldehyde cyanohydrin and2-amino-m-cresol using Method C2.

1-Hydroxy-1-(5-methylbenzoxazol-2-yl)-9(Z)-octadecene: This material wasprepared in 79% yield from oleyl aldehyde cyanohydrin and2-amino-p-cresol using Method C2.

1-Hydroxy-1-(6-methylbenzoxazol-2-yl)-9(Z)-octadecene: This material wasprepared in 59% yield from oleyl aldehyde cyanohydrin and6-amino-m-cresol using Method C2.

1-Hydroxy-1-(7-methylbenzoxazol-2-yl)-9(Z)-octadecene: This material wasprepared in 53% yield from oleyl aldehyde cyanohydrin and6-amino-o-cresol (Bisarya, S. C., et al., (1993) Synth. Commun. 23,1125-1137) using Method C2.

1-(2-Benzoxazolyl)-1-oxo-9(Z)-octadecene (23): This material wasprepared in 67% yield from the alcohol using the procedure described for18.

1-(4-Methylbenzoxazol-2-yl)-1-oxo-9(Z)-octadecene (25): This materialwas prepared in 81% yield from the alcohol using the procedure describedfor 18.

1-(5-Methylbenzoxazol-2-yl)-1-oxo-9(Z)-octadecene (26): This materialwas prepared in 35% yield from the alcohol using the procedure describedfor 18.

1-(6-Methylbenzoxazol-2-yl)-1-oxo-9(Z)-octadecene (27): This materialwas prepared in 66% yield from the alcohol using the procedure describedfor 18.

1-(7-Methylbenzoxazol-2-yl)-1-oxo-9(Z)-octadecene (28): This materialwas prepared in 74% yield from the alcohol using the procedure describedfor 18.

1-(2-Benzimidazolyl)-1-hydroxy-9(Z)-octadecene (58): This material wasprepared in 54% yield from oleyl aldehyde cyanohdrin and1,2-phenylenediamine using the procedure described as Method C2. 58; mp109-110 C.

1-(2-Benzimidazolyl)-1-oxo-9(Z)-octadecene (21): This material wasprepared in 73% yield from the alcohol using the procedure described for18.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)-9(Z)-octadecene (59). MethodC3: A solution of anhydrous EtOH (0.52 mL, 8.86 mmol, 20.6 equiv) inCHCl₃ (1 mL) at 0 C was treated with acetyl chloride (0.50 mL, 7.03mmol, 16.4 equiv) followed by a solution of oleyl aldehyde cyanohydrin(126.3 mg, 0.430 mmol, 1 equiv) in CHCl₃ (1.5 mL). The reaction mixturewas allowed to warm to 25 C and stirred for 13 h. The solvent wasevaporated to afford the imidate as a white solid which was used withoutfurther purification.

A solution of the imidate in dry EtOCH₂CH₂OH (1.5 mL) was treated with2-amino-3-hydroxypyridine (48.0 mg, 0.436 mmol, 1.01 equiv). Thereaction mixture was heated at 130 C for 6 h. The reaction mixture wasevaporated and the residue was dissolved in EtOAc/Et₂O (2:1) (50 mL) and1 N aqueous NaOH (10 mL), and washed with saturated aqueous NaCl (20mL). The organic layers were dried (Na₂SO₄), filtered, and evaporated.Chromatography (SiO₂, 1.5 18 cm, 3% MeOHCH₂Cl₂ and then SiO₂, 1.5 18 cm,66% EtOAchexanes) afforded the product alcohol 59 (27.3 mg, 16%) as apale brown oil.

1-Hydroxy-1-(oxazolo[4,5-c]pyridin-2-yl)-9(Z)-octadecene: This materialwas prepared in 0.7% yield from oleyl aldehyde cyanohydrin and3-amino-4-hydroxypyridine using Method C2.

1-Hydroxy-1-(oxazolo[4,5-d]pyridin-2-yl)-9(Z)-octadecene: This materialwas prepared in 1.7% yield from oleyl aldehyde cyanohydrin and4-amino-3-hydroxypyridine using Method C3.

1-Hydroxy-1-(oxazolo[4,5-e]pyridin-2-yl-9(Z)-octadecene: This materialwas prepared in 2% yield from oleyl aldehyde cyanohydrin and3-amino-2-hydroxypyridine using Method C2.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-9(Z)-octadecene (29): This materialwas prepared in 76% yield from the alcohol using the procedure describedfor 18.

1-(Oxazolo[4,5-c]pyridin-2-yl)-1-oxo-9(Z)-octadecene (30): This materialwas prepared in 87% yield from the alcohol using the procedure describedfor 18.

1-(Oxazolo[4,5-d]pyridin-2-yl)-1-oxo-9(Z)-octadecene (31): This materialwas prepared in 28% yield from the alcohol using the procedure describedfor 18.

1-(Oxazolo[4,5-e]pyridin-2-yl)-1-oxo-9(Z)-octadecene (32): This materialwas prepared in 36% yield from the alcohol using the procedure describedfor 18.

1-(2-Benzoxazolyl)-1-oxo-octadecane (37): A solution of 23 (5.2 mg,0.0136 mmol) in MeOH (0.5 mL) was combined with 10% PdC (2.2 mg) underN₂. The atmosphere was purged with H₂ and the reaction mixture wasstirred at 25 C for 10 min. The reaction mixture was filtered andevaporated. Chromatography (SiO₂, 1.5 10 cm, 50% CH₂Cl₂hexanes) afforded37 (2.4 mg, 46%) as a white solid; mp 71-72 C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-octadecane (34): This material wasprepared in 72% yield from 29 using the procedure described for 37.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)-9(E)-octadecene: This materialwas prepared in 15% overall yield from 9(E)-octadecenal and2-amino-3-hydroxypyridine using Method C3.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-9(E)-octadecene (33): This materialwas prepared in 60% vield from the alcohol using the procedure describedfor 18. 33; mp 49-51° C.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)-5(Z),8(Z),11(Z),14(Z)-eicosatetraene:This material was prepared in 10% yield from arachidonyl aldehyde and2-amino-3-hydroxypyridine using Method C3.

1-(Oxazolo[4,5-b]pyridin-2-yl)arachidonaldehyde (38): This material wasprepared in 31% yield from the alcohol using the procedure described for18.

1-(Oxazolo[4,5-c]pyridin-2-yl)arachidonaldehyde (39):

1-Hydroxy-1-(oxazolo[4,5-c]pyridin-2-yl)-5(Z),8(Z),11(Z),14(Z)-eicosatetraenewas prepared from arachidonyl aldehyde and 3-amino-4-hydroxypyridineusing method C3. This unstable alcohol was immediately oxidized usingthe procedure described for 18 to give 39.

1-(Oxazolo[4,5-d]pyridin-2-yl) arachidonaldehyde (40):

1-Hydroxy-1-(oxazolo[4,5-d]pyridin-2-yl)-5(Z),8(Z),11(Z),14(Z)-eicosatetraenewas prepared from arachidonyl aldehyde and 4-amino-3-hydroxypyridineusing method C3. This unstable alcohol was immediately oxidized usingthe procedure described for 18 to give 40.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)hexadecane. Method C4: Asolution of hexadecanal (160.0 mg, 0.666 mmol, 1 equiv) in THF (5.7 mL)and H₂O (6.2 mL) was treated with KCN (446.1 mg, 6.85 mmol, 10.3 equiv).The reaction mixture was stirred at 25° C. for 70 h. H₂O (20 mL) andEt₂O (20 mL) were added to the reaction mixture. The mixture wasextracted with Et₂O (3 20 mL) and washed with saturated aqueous NaHCO₃(20 mL) and saturated aqueous NaCl (20 mL). The extracts were dried(Na₂SO₄), filtered, and evaporated to afford the cyanohydrin (162.0 mg,91%) as a white solid which was used without further purification.

A solution of anhydrous EtOH (0.72 mL, 12.1 mmol, 20.0 equiv) in CHCl₃(1.5 mL) at 0 C was treated with acetyl chloride (0.69 mL, 9.70 mmol,16.0 equiv) followed by a solution of the cyanohydrin (162.0 mg, 0.606mmol, 1 equiv) in CHCl₃ (3 mL). The reaction mixture was allowed to warnto 25 C and stirred for 20.5 h. The solvent was evaporated to afford theimidate as a white solid which was used without further purification.

A solution of the imidate in dry 2-ethoxyethanol (2.5 mL) was treatedwith 2-amino-3-hydroxypyridine (66.6 mg, 0.605 mmol, 1.0 equiv). Thereaction mixture was heated to 125 C for 6.5 h. The reaction mixture wasevaporated and the residue was dissolved in EtOAc/Et₂O (2:1) (60 mL) and1 N aqueous NaOH (10 mL), and washed with saturated aqueous NaCl (20mL). The organic layers were dried (Na₂SO₄), filtered, and evaporated.Chromatography (SiO₂, 1.5 22 cm, 3% MeOHCH₂Cl₂ and then SiO₂, 1.5 20 cm,66% EtOAchexanes) afforded the alcohol (24.7 mg, 11%) as a pale brownsolid; mp 59-61° C.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)ethane: This material wasprepared acetaldehyde using the Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)pentane: This material wasprepared from pentanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)hexane: This material wasprepared from hexanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)heptane: This material wasprepared from heptanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)octane: This material wasprepared from octanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)decane: This material wasprepared from decanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)dodecane (60): This material wasprepared from dodecanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)tetradecane: This material wasprepared from tetradecanal using Method C4: mp 52-54° C.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexane: This materialwas prepared from 6-phenylhexanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)-7-phenylheptane: This materialwas prepared from 7-phenylheptanal using Method C4.

1-Hydroxy-1-(oxazolo[4,5-b]pyridin-2-yl)-8-phenyloctane: This materialwas prepared from 8-phenyloctanal using Method C4.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-hexadecane (42): This material wasprepared in 65% yield from the alcohol using the procedure described for18. 42; mp 77-78° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-ethane (50): This material wasprepared in 75% yield from the alcohol using the procedure described for18. 50; mp 103-105° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-pentane (49): This material wasprepared in 64% yield from the alcohol using the procedure described for18. 49; mp 35-37° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-hexane (48): This material wasprepared in 70% yield from the alcohol using the procedure described for18. 48; mp 51-53° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-heptane (47): This material wasprepared in 64% yield from the alcohol using the procedure described for18. 47; mp 52-53° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-octane (46): This material wasprepared in 54% yield from the alcohol using the procedure described for18. 46; mp 60-61° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-decane (45): This material wasprepared in 61% yield from the alcohol using the procedure describedabove for compound 18. 45; mp 60-62° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-dodecane (44): This material wasprepared in 94% yield from the alcohol using the procedure described for18. 44; mp 68-69° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-tetradecane (43): This material wasprepared in 72% yield from the alcohol using the procedure describedabove for compound 18. 43; mp 73-74° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-6-phenylhexane (53): This materialwas prepared in 78% yield from the alcohol using the procedure describedfor 18. 53; mp 61-63° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-7-phenylheptane (54): This materialwas prepared in 74% yield from the alcohol using the procedure describedfor 18. 54; mp 60-61° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-1-oxo-8-phenyloctane (55): This materialwas prepared in 72% yield from the alcolol using the procedure describedabove for compound 18. 55; mp 70-73° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)octadecane (61): This material wasprepared in 30% yield from 1-cyanooctadecane (Mangold, H. K., et al.,(1976) Chem. Phys. Lipids 17, 176-181) and 2-amino-3-hydroxypyridineusing Method C3: mp 84-85° C.

1-(Oxazolo[4,5-b]pyridin-2-yl)-9(Z)-octadecene (62): This material wasprepared in 25% yield from 1-cyano-9(Z)-octadecene (Baumann, W. J. etal., (1968) J. Lipid Res. 9, 287) and 2-amino-3-hydroxypyridine usingMethod C3.

1. An inhibitor of fatty acid amide hydrolase represented by thefollowing formula:A-B-C wherein: A is an α-keto heterocyclic pharmacophore for inhibitingthe fatty acid amide hydrolase; B is a chain for linking A and C, saidchain having a linear skeleton of between 3 and 9 atoms selected fromthe group consisting of carbon, oxygen, sulfur, and nitrogen, the linearskeleton having a first end and a second end, the first end beingcovalently bonded to the α-keto group of A, with the following proviso:if the first end of said chain is an α-carbon with respect to the α-ketogroup of A, then the α-carbon is optionally mono- or bis-functionalizedwith substituents selected from the group consisting of fluoro, chloro,hydroxyl, alkoxy, trifluoromethyl, and alkyl; and C is an activityenhancer for enhancing the inhibition activity of said α-ketoheterocyclic pharmacophore, said activity enhancer having at least onep-unsaturation situated within a p-bond containing radical selected froma group consisting of aryl, alkenyl, alkynyl, and ring structures havingat least one unsaturation, with or without one or more heteroatoms, saidactivity enhancer being covalently bonded to the second end of thelinear skeleton of B, the p-unsaturation within the p-bond containingradical being separated from the α-keto group of A by a sequence of noless than 4 and no more than 9 atoms bonded sequentially to one another,inclusive of said linear skeleton.
 2. An inhibitor of fatty acid amidehydrolase according to claim 1 wherein said α-keto heterocyclicpharmacophore is represented by the formula:

wherein “het” is selected from the following group:


3. An inhibitor of fatty acid amide hydrolase according to claim 2wherein “het” is selected from the following group:


4. An inhibitor of fatty acid amide hydrolase according to claim 3represented by the following structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, fluoro, chloro, hydroxyl, alkoxy, trifluoromethyl, andalkyl; and n” is an integer between 2 and
 7. 5-12. (canceled)